Chemical simulation of hypoxia in donor cells improves development of somatic cell nuclear transfer‐derived embryos and increases abundance of transcripts related to glycolysis

Abstract

To improve efficiency of somatic cell nuclear transfer (SCNT), it is necessary to modify differentiated donor cells to become more amendable for reprogramming by the oocyte cytoplasm. A key feature that distinguishes somatic/differentiated cells from embryonic/undifferentiated cells is cellular metabolism, with somatic cells using oxidative phosphorylation (OXPHOS) while embryonic cells utilize glycolysis. Inducing metabolic reprogramming in donor cells could improve SCNT efficiency by priming cells to become more embryonic in nature before SCNT hypoxia inducible factor 1‐α (HIF1‐α), a transcription factor that allows for cell survival in low oxygen, promotes a metabolic switch from OXPHOS to glycolysis. We hypothesized that chemically stabilizing HIF1‐α in donor cells by use of the hypoxia mimetic, cobalt chloride (CoCl 2 ), would promote this metabolic switch in donor cells and subsequently improve the development of SCNT embryos. Donor cell treatment with 100 µM CoCl 2 for 24 hr preceding SCNT upregulated messenfer RNA abundance of glycolytic enzymes, improved SCNT development to the blastocyst stage and quality, and affected gene expression in the blastocysts. After transferring blastocysts created from CoCl 2 ‐treated donor cells to surrogates, healthy cloned piglets were produced. Therefore, shifting metabolism toward glycolysis in donor cells by CoCl 2 treatment is a simple, economical way of improving the in vitro efficiency of SCNT and is capable of producing live animals.

Treatment of Donor Cells with Oxidative Phosphorylation Inhibitor CPI Enhances Porcine Cloned Embryo Development

“…In the present study, the blastocyst rate of SCNT embryos cloned from CPI-treated PFFs was significantly higher compared to the NC group treatment of PFFs with 100 µM CoCl 2 (a glycolysis inducer) and also resulted in a significantly higher blastocyst rate and blastocyst cell number in subsequent SCNT embryos, compared to NC [31]. Similarly, treatment of PFFs with 10 µM PS-48, another glycolysis inducer, led to significantly higher cleavage and blastocyst rates in subsequent cloned embryos compared to NC [30].…”

“…Furthermore, treating buffalo fetal fibroblasts with glycolysis inducer PS48 resulted in the increased production of intracellular lactic acid and a consequential enhancement in the development of cloned embryos [30]. The treatment of porcine fibroblasts with 100 µM CoCl 2 (a glycolysis inducer) led to the increased expression of glycolytic enzyme genes and the enhanced development of cloned embryos [31].…”

Production of Pigs From Porcine Embryos Generated in vitro

“…Different abnormalities have been detected in piglets derived by the IVP system and manipulation procedures, although predominantly the reports are related to cloned piglets. High rates of stillbirths have been noted for pregnancies carrying clones (Estrada et al, 2007;Schmidt et al, 2015;Ao et al, 2017;Cecil et al, 2020), and increased mortality within the first few days after farrowing is often observed with cloned pigs (Ao et al, 2017). Birth weight may be a predictor of survival probability for cloned piglets as those that died within 4 days after birth weighed ∼28% less than those that survived for more than 4 days (Ao et al, 2017).…”

Manipulating the Epigenome in Nuclear Transfer Cloning: Where, When and How

“…Hypoxia inducible factor 1 subunit alpha (HIF1A), a transcription factor that allows for cell survival at low oxygen tension, promotes a metabolic switch from somatic cell specific oxidative phosphorylation to glycolysis used by early embryos [ 119 , 120 ]. Stabilization of HIF1A by treatment of donor cells with cobalt chloride (CoCl 2 ) upregulated mRNA abundances of glycolytic enzymes and improved development of porcine cloned embryos to the blastocyst stage [ 121 ]. Shifting the metabolism of donor cells toward glycolysis can thus be a simple way for improving cloning efficiency.…”